November 1, 2017 (Vol. 37, No. 19)

Applying Acoustic Technology to Reduce Shear in Cell-Therapy Processing

As the number of advanced treatments involving cell and gene therapies increases, research has shown that traditional methods to process cells for therapy production have high shear forces. Shear can significantly impact cell membranes and even the markers needed to generate the desired clinical response, causing a barrier to innovation for developers and manufacturers of these therapies.

In this article, we discuss the urgent need for cell handling and sorting technologies that minimize impact on cells, with a brief look into traditional approaches and the challenges they present. We suggest 3D acoustic wave separation technology as a solution to these challenges, including an overview of how it works, where it has been applied so far, and possible future applications.

Rapidly Developing Cell Therapy Market

Early-phase clinical trial results for many types of cell therapies are showing real potential to treat—and in some cases, cure—diseases that have been historically been difficult to address with traditional small-molecule and newer biologic drugs. Significant funds are being directed toward cell therapy development and commercialization efforts by both pharmaceutical companies and investors.

According to market research firm Roots Analysis, more than 60 industry players and 50 academic institutes/nonprofit organizations are actively contributing to the field of cell-therapy manufacturing, with more than 500 cell-based therapy candidates currently in different stages of clinical development and at least 1,000 active clinical studies underway around the world.1 The firm predicts the market for advanced therapy medicinal products (ATMPs) based on immune cells (i.e., T cells, Tregs, dendritic cells, tumor cells, NK cells) and stem cells (i.e., adult, human, embryonic [ESCs], induced pluripotent [iPSCs]) will grow at an annualized rate of ~42% over the next ten years, reaching a value of $4 billion in 2027.

Impact of Shear Forces on Cell Properties

The behavior of cells within living tissue is influenced by both the chemical and physical conditions within their microenvironments. For instance, growth factors and other biochemicals direct cellular binding and signaling activities, while physical forces affect the cellular structure and other cell functions.2,3 Similarly, cell microenvironments during manufacturing impact the structure and function—and ultimately the safety and efficacy—of cellular therapies. Bioprocess forces during cell culture, purification (typically centrifugation and/or filtration), and transfer must be carefully considered when designing manufacturing processes.

While limited studies have been conducted on cellular therapies, the effects of process forces have been widely demonstrated. The impact of force exerted during bioprocessing depends on the cell type and variations within cell populations. Different forces during different unit operations (cell culture, centrifugation, etc.) also affect cells in diverse ways. This includes damage to cell membranes, but also reduction in cell-expansion performance and the production of key biochemicals have been shown to occur in response to the shear forces experienced during bioprocessing.2 These results are not surprising given that mammalian cells used for cell culture of biologic drug substances have previously been shown to be negatively affected by exposure to increasing shear forces.4

Issues with Centrifugation for Cell Washing and Purification

Following cell culture and harvesting of the live cells for cell therapy products, the cells are often in a dilute bioprocess solution that must be concentrated. Washing is also necessary to remove residual media, serum, other ingredients, and any contaminants generated during cell culture. Because the cells at this stage exist without nutrients in atypical conditions, these steps must be performed within a few hours to prevent a reduction in quality.

At the clinical scale, manual/batch centrifugation is typically performed at this stage, often repeatedly, to yield a solid pellet of cells with the desired purity. The pellet is then resuspended to generate the concentrated and washed solution for final formulation and cryopreservation. The shear forces involved in centrifugation may negatively impact the quality of the cells, while the time required for repeated centrifugation can also lead to cell degradation. In addition, pelletization can cause unwanted aggregation.

Furthermore, manual centrifugation is performed in an open environment, which is problematic in the production of commercial cell therapies, given that they cannot be sterilized at the end of the manufacturing process. Closed systems that ensure aseptic processing are essential. The need for repeated cycles can lead to processes that are too lengthy. Compared with automated systems, manual processes are also susceptible to human error and suffer from limited process control and reduced product consistency. Furthermore, such processes are not readily scalable to produce the larger quantities of cell therapy drug products required for late-phase clinical trials and ultimately commercialization.5

There are some automated, closed systems for cell concentration and washing, including automated counter-flow centrifugation, and alternating tangential-flow and tangential-flow filtration devices.6,7 Spinning membrane filtration is also under development. However, these systems still have drawbacks: Cell damage may occur due to the shear forces involved; scalability may still be an issue; and if not available as single-use technologies, pre-sterilization is required. 

Acoustic Wave Separation as an Alternative Technology

FloDesign Sonics has developed a technology for continuous cell concentration and washing based on acoustophoretic separation technology.8–10 A single-use (gamma-irradiated) system has been designed specifically for the manufacture of cell-based therapies and is currently available for process development.

Importantly, acoustophoretic separation technology for the continuous clarification of monoclonal antibodies and other therapeutic proteins is already being commercialized through a limited use cell-process license by FloDesign Sonics to Pall (the license does not cover full cell and gene therapy applications).11,12 With the single-use CadenceTM acoustic separator from Pall, continuous removal of cells is achieved in a closed system without centrifugation, allowing for clarification in a small operating footprint without any detectable impact on either the cells or the product itself. High product yields are obtained with predictable and reproducible performance over a wide range of cell densities at both laboratory and commercial scales.

In FloDesign Sonics’ system, designed for the downstream processing of cell therapies, cells from a cell suspension are trapped and held in an acoustic radiation force field comprising standing acoustic waves generated by a transducer. The fluid flows through the device, which retains 90% or more of the cells. The trapped cells can then be washed with the desired buffer solution one or more times, displacing the original cell culture fluid. The entire system is enclosed and the cells are not exposed to any undesirable shear forces.

Controlled by a LabVIEW program, the system is highly automated, allowing control of up to five channels at once. Operating parameters are entered into an easy-to-use interface, and the program then controls the flow rates, flow paths, power levels, and draw rates.

The process development–scale device is sized for feed volumes of 250 mL to 3 L. The current element can process approximately 2 L per hour with a capacity of approximately 40 billion cells, affording a final concentrated volume of approximately 6–100 mL.

Concentration factors of 10 to 200X have been demonstrated. Complete flushing/washing of various buffer and media from the system after three system volumes was confirmed using Molecular Devices’ SpectraMax (via measurement of absorbance at two different wavelengths). A cell-retention efficiency of 90% is also achieved without any effect on cell viability.

Cell density reduction of Jurkat T-cells [ATCC, TIB-152] (diameter of 11–14 µm) was evaluated using a Beckman Coulter Vi-CELL XR at various test conditions during more than 80 trials at FloDesign Sonics and has been demonstrated to be >80%. In recent trials at a customer site using primary cultures of T cells for concentration and a 2-volume wash, cell viabilities of >90% were obtained, and the viability never decreased from the feed to the final concentrate volume.

A scaled prototype will handle higher flow rates and cell densities. The acoustic element and collector would be larger to increase the cell-holding capacity and allow processing of a larger volume within the same timeframe. The system can handle feed volumes up to 5–10 L and process feed concentrations of 1 to 40 million cells/mL. It has a total cell volume capacity of 150 billion cells. Final conrozemcentrate volumes range from 50 to 500 mL.


The acoustic cell-processing platform offers a robust cell concentration and washing unit operation for cell therapy applications. Acoustophoretic separation technology from FloDesign Sonics addresses the pain points for concentration and washing of cell therapies, including low cell recovery, long wash cycles with the use of large buffer volumes, open-process manual manipulations, complexity, and lack of scalability. Technology for handling the full spectrum of feed volumes, cell types, and cell concentration processes expected during the development and commercialization of cell therapies is under development. 

John Rozembersky is vice president of biopharm technologies; Rui Tostoes, Ph.D., is senior process engineer; and Chris Leidel ([email protected]), is vice president of business development at FloDesign Sonics.

1. Roots Analysis, “Cell Therapy Manufacturing Market, 2017-2027,” (March 3, 2017), accessed October 4, 2017.
2. D. Brindley et al., “Bioprocess Forces and Their Impact on Cell Behavior: Implications for Bone Regeneration Therapy,” J. Tissue Eng., doi:10.4061/2011/620247 (August 2011).
3. A. Salameh and S. Dhein, “Effects of Mechanical Forces and Stretch on Intercellular Gap Junction Coupling,”  Biochim. Biophys. Acta (BBA)-Biomembranes 1828(1), 147–156 (January 2014).
4. B. Vickroy et al., “Modeling Shear Damage to Suspended CHO Cells during Cross-Flow Filtration,” Biotech. Prog. 23(1), doi:10.1021/bp060183e (2007).
5. Y. Eylon et al., “Foundation Elements for Cell Therapy Smart Scaling,” Bioproc. Int., (April 14, 2015), accessed October 4, 2017.
6. A.C. Schnitzer et al., “Bioprocessing of Human Mesenchymal Stem/Stromal Cells for Therapeutic Use: Current Technologies and Challenges,” Biochem. Eng. J. 108, 3–13 (2016).
7. Q.A. Rafiq and F. Masri, “Downstream Processing for Cell-Based Therapies,” BioPharm Int. 30(4), 22–26 (April 19, 2017).
8. B. Lipkens et al., “A Novel Acoustic Cell Processing Platform for Cell Concentration and Washing,” ISCT annual meeting poster presentation, London, UK (May 3–6, 2017).
9. J.J. Rozembersky et al., “LB27—A Novel Scalable Acoustic Cell Processing Platform for Cell Concentration and Washing,” Cytotherapy 18(5), supplement p. e17 (May 2017).
10. J. Dionne, B. Dutra, and B. Lipkens, “A Novel Acoustic Cell Processing Platform for Cell Concentration and Washing,” J. Acoust. Soc. Am. 141, 3741 (2017), doi:.
11. Genetic Engineering and Biotechnology News (GEN), “Pall Acquires Innovative Acoustic Wave Separation Technology”, accessed October 4, 2017.
12. P.R. Levison, “Acoustic Wave Separation—A Scalable Disruptive Technology for Continuous Clarification of Fed Batch Cell Culture Prior to Capture Chromatography”, accessed October 4, 2017.

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